Thrombin‐dependent calcium signalling in single human erythroleukaemia cells

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Abstract
1. A combination of single cell fluorescence and patch clamp techniques were used to study the mechanisms underlying thrombin-evoked Ca2+ signals in human erythroleukaemia (HEL) cells, a leukaemic cell line of platelet-megakaryocyte lineage. 2. Thrombin caused a transient increase in intracellular Ca2+ ([Ca2+]i), consisting of both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Mn2+ quench studies indicated that the thrombin-evoked divalent cation-permeable pathway was activated during, but not prior to, release from internal stores. 3. Thapsigargin (1 microM) irreversibly released internal Ca2+ from the same store as that released by thrombin and continuously activated a Ca(2+)-influx mechanism. The amplitude of the thrombin- and thapsigargin-induced Ca2+ influx displayed a marked single cell heterogeneity which showed no correlation with the size of the store Ca2+ transient. 4. In whole-cell patch clamp recordings, both thrombin and thapsigargin evoked an inwardly rectifying Ca2+ current which developed with little or no increase in current noise, showed no reversal in the voltage range -110 to +60 mV and was blocked by 1 mM Zn2+. The apparent divalent cation permeability sequence of this pathway was Ca2+ > > Ba2+ > Mn2+, Mg2+. The thapsigargin-evoked current density at -100 mV varied between 0.42 and 2.1 pA pF-1 in different cells. Thrombin failed to activate additional Ca2+ current if it was added after the thapsigargin-induced inward current had fully developed. 5. These studies indicate that thrombin activates Ca2+ influx in HEL cells entirely via a Ca(2+)-store-release-activated Ca2+ current (Icrac) rather than via receptor-operated or second messenger-dependent Ca2+ channels. The level of expression of Icrac appears to be a major factor in determining the duration of the thrombin-evoked [Ca2+]i response and therefore represents a means by which cells can exert control over [Ca2+]i-dependent events.